Cancer lines contaminated

Three of 13 established esophageal adenocarcinoma (EAC) cell lines are not EAC after all. Instead, the lines -- which have led to two clinical trials, more than 100 publications and 11 US patents -- represent three different cancer types altogether, a study published online today (January 14) in the Journal of the National Cancer Institute (JNCI) found.
Histopathologic image illustrating welldifferentiated squamous cell carcinomain the excisional biopsy specimenImage: Wikimedia commons"

By Jef Akst | January 14, 2010

Three of 13 established esophageal adenocarcinoma (EAC) cell lines are not EAC after all. Instead, the lines -- which have led to two clinical trials, more than 100 publications and 11 US patents -- represent three different cancer types altogether, a study published online today (January 14) in the Journal of the National Cancer Institute (JNCI) found.

"It's a startling and somewhat alarming finding that the cell lines we thought represented specific entities actually do not," said cancer biologist linkurl:Ezra Cohen,;http://www.uchospitals.edu/physicians/ezra-cohen.html who was heading up a clinical trial based partially on experiments conducted with one of the three contaminated EAC cell lines at the time these results became known to the field a few months ago. "If you're going to do studies focusing on [a specific] disease, you probably want to do it on cell lines from that disease -- otherwise the findings might not have the same implications."
Misidentification of cell lines is not a new problem. In the 1970s, for example, cytogenetic analyses revealed widespread contamination by HeLa cells, which prompted some of the first guidelines for cell line quality control. Since that time, molecular techniques have advanced to allow scientists to verify their cell lines with increased resolution and at a much faster pace, which has in turn led to the identification of more contaminated lines.
Two years ago, molecular biologist linkurl:Winand Dinjens;http://www.erasmusmc.nl/pathologie/research/dinjens/166240/?lang=nl&view=Print of the Josephine Nefkens Institute, Erasmus MC, University Medical Center Rotterdam, The Netherlands and his colleagues made the linkurl:unexpected discovery;http://cancerres.aacrjournals.org/cgi/content/abstract/67/17/7996 that one of the cell lines they believed to be derived from EAC actually shared the same genotype as four esophageal squamous cell carcinoma cell lines. "That result prompted us to try to verify all EAC cell lines," Dinjens said.
With the cooperation of the investigators who had originally derived the EAC cell lines, Dinjens' team was able to trace the pathology archives -- where the tissues from the patients are still present -- for 13 of 14 established EAC cell lines. The researchers then used genotyping assays to compare the DNA retrieved from those tissues to the DNA from the established cell lines. Three of the 13 cell lines did not match up.
"We were surprised and kind of uncomfortable" with these results, Dinjens said. "Two of these three lines are by far the most used cell lines in this research field."
All three contaminated lines, SEG-1, BIC-1, and SK-GT-5, could be traced back to other cell lines -- large cell lung cancer, colorectal adenocarcinoma, and gastric fundus carcinoma, respectively.
How the contamination occurred is unclear, but there are two most likely explanations, Dinjens said. First, it could be a case of simply mislabeling the cultures. If this happens soon after the lines are created, "then all the offspring [lines] are incorrect," he said. Alternatively, contamination could occur in the lab during the derivation of the lines. Because cells from a small tumor specimen initially grow very slowly, Dinjens explained, a single droplet of an established cell line that grows much faster will eventually take over the whole culture. "You still think you have two cell lines, but you only have one," he said.
But while three of the 13 EAC cell lines were contaminated, the other 10 proved to be authentic -- not a bad percentage considering some estimates suggest that up to one third of all cell lines have a different origin than is supposed, said cancer biologist linkurl:Robert Shoemaker;http://spheroid.ncifcrf.gov/spheroid/stb/staff/shoemaker/shoemaker.cfm of the National Cancer Institute in Maryland, who wrote an accompanying editorial to the JNCI report. "They made a good start by identifying 10 bona fide [EAC] cell lines," he said.
Also, "cancer is a heterogeneous disease," Shoemaker said -- "using one cell line of any type as a model is just very weak science." Indeed, most therapies are not derived on the basis of in vitro experiments on a single cell line. The identification of these three contaminated lines therefore does not necessarily negate all the research that has used the lines.
Cohen's linkurl:clinical trial,;http://www.clinicaltrials.gov/ct2/show/NCT00619242 for example, was not based solely on data from that one cell line, Cohen said, and at the time the news of the contaminated lines was released, "we didn't feel like it was enough of an impact to suspend the study." (The trial has since been closed for unrelated reasons, including slow patient recruitment and mild adverse side effects.)
Furthermore, many cancer therapies target pathways that are commonly affected in cancer. The linkurl:other clinical trial;http://www.clinicaltrials.gov/ct2/show/NCT00310895?term=GRN163L&rank=2 that is at least partially based on results from one of the three contaminated EAC cell lines, for example, targets telomerase, which is "believed to be pretty much ubiquitous in cancer," Shoemaker said. Thus, the trial "could probably have been justified" without the EAC cell line experiments.
Importantly, the 10 authenticated EAC lines identified in this study, as well as their genotyping information, will be put into publically available cell line repositories in the US, Europe, and Japan. Still, "it's important to have standard operating procedure in place to make sure that the cell lines they think they are," said Cohen, who did not participate in the JCNI study. "As a scientific community, we are becoming much more aware of [contamination issues], and I think it's an important process to go through."
**__Related stories:__***linkurl:Fly study retracted;http://www.the-scientist.com/blog/display/56108/ [20th October 2009]*linkurl:A case of mistaken identity;http://www.the-scientist.com/news/display/55013/ [16th September 2008]*linkurl:Keeping Tabs on Cultured Cells;http://www.the-scientist.com/article/display/15310/ [14th March 2005]

Comments

This has been an ongoing problem for a long time(see references). Unfortunately, it doesn't even address the issue of how poorly even the correctly identified cell lines reflect the original tumors. After all, many of the widely used lines were grown on tissue culture plastic in fetal bovine serum for a year before they were even submitted to a repository. Sure, they're still cells, but are hardly more representative of the original tumors than are yeast cells. This is not to say cell cultures aren't useful cancer research tools, but rather that too many folks have been using the same 'cell' tools their father used. It's like driving a Chevette when you've got a Prius in the garage! Once there are hundreds or even more publications using a particular cell line, no one wants to go back and address the issue. How about going forward, with new cell cultures established using what we've learned over the past 40 or 50 years?!\n\nMDA-MB-435 and M14 Cell Lines: Identical but not M14 Melanoma? Ann F. Chambers. Cancer Res 2009; 69: (13): 5292-5293, 2009.\n\nCurbing rampant cross-contamination and misidentification of cell lines. Roland M. Nardone. BioTechniques 45(3): 221-227, 2008.\n\nDeoxyribonucleic Acid Profiling Analysis of 40 Human Thyroid Cancer Cell Lines Reveals Cross-Contamination Resulting in Cell Line Redundancy and Misidentification. Rebecca E. Schweppe et al. J Clin Endocrinol Metab 93(11):4331?4341, 2008.\n\nThe costs of using unauthenticated, over-passaged cell lines: how much more data do we need? Peyton Hughes et al. BioTechniques 43:575-586, 2007. \n\nCases of Mistaken Identity. For decades, biologists working with contaminated or misidentified cell lines have wasted time and money and produced spurious results; journals and funding agencies say it's not their job to solve this problem. Rhitu Chatterjee. News Focus, Science 315: 928-931, 2007. \n\nCell Line Cross-Contamination: How Aware Are Mammalian Cell Culturists of the Problem and How To Monitor It? Gertrude Case Buehring, Elizabeth A. Eby, And Michael J. Eby. In Vitro Cell. Dev. Biol.?Animal 40:211?215, July?August 2004. \n\nInterspecies Contamination of the Km3 Cell Line: Implications for Cd63 Function in Melanoma Metastasis. Gregory W. Moseley et al. Int. J. Cancer: 105, 613?616 , 2003. \n\nBreast cancer cell lines: friend or foe? Sarah E Burdall et al. Breast Cancer Res 5:89-95, 2003. \n\nMix-ups and mycoplasma: the enemies within. Hans G. Drexler et al. Leukemia Research 26: 329?333, 2002. \n\nShort tandem repeat profiling provides an international reference standard for human cell lines. John R. Masters et al. Proc. Natl. Acad. Sci. USA 98(14):8012?8017, 2001. \n

An interesting and important finding underlining that this type of cancer can have a complex composition on the cellular level. Maybe researchers should rather consider that uniform mutation patterns in cancer cells from biopsy samples might be rather exceptions, and that a certain variety of mutations and their concerted combinations exists. \n\nA big problem as mentioned before are artefacts caused by tissue culture under uniform conditions for long time periods - what mutations arise after the biopsy time point and how does this influence the cancer cell subpopulation variety? An artificial cell variety in the maintained cancer cell population might lead to misconclusions with respect to effects of experimental drugs, which work in tissue cultures in vitro, but might maybe logically fail in vivo.\n\nWhat mutations and cell subpopulations are present in the original cancer samples, and how can this natural variety be kept under experimental culture conditions?\n\nBypassing the weaknesses of this question could help cancer research a lot.